Submitochondrial Location and Some Properties of NAD+- and NADP+-Linked Malate Dehydrogenase inEuglena

Isegawa, Yuji; Nakano, Yoshiaki; Kitaoka, Shôzaburô

doi:10.1080/00021369.1984.10866181

Cited by 2 publications

(5 citation statements)

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“…The purified mitochondrial fraction was recovered at the interfase of 30 Percoll, and it was washed and resuspended in the same buffer used for suspending crude mitochondria. The obtained mitochondria were completely free from other subcellular fractions and were intact [25]. A suspension of purified mitochondria containing 2 mM dithiothreitol was sonicated (10 kHz, 10 s) and centrifuged at 10000 x g for 10min to remove unbroken mitochondria, and the supernatant was centrifuged at I00000 x g for 60min to separate soluble and membrane fractions.…”

Section: Orgunisms and Chemicalsmentioning

confidence: 99%

Fatty acid synthesis in mitochondria of Euglena gracilis

Inui

Miyatake

Nakano

et al. 1984

European Journal of Biochemistry

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A malonyl-CoA-independent fatty acid synthetic system, different from the systems in other subcellular fractions, occurred in mitochondria of Euglena gracilis. The system had ability to synthesize fatty acids directly from acetylCoA as both primer and C , donor using NADH as an electron donor. Fatty acids were synthesized by reversal of fi-oxidation with the exception that enoyl-CoA reductase functioned instead of acyl-CoA dehydrogenase in degradation system. A fairly high activity of enoyl-CoA reductase was found on various enoyl-CoA substrates (C, -C,,) with NADH or NADPH. Three species of enoyl-CoA reductase, distinct from each other by their chain-length specificity, were found in Euglena mitochondria, and one of them was highly specific for crotonyl-CoA. It is also discussed that the mitochondrial fatty-acid synthetic system contributes to wax ester fermentation, the anaerobic energy-generating system found in the organism.We have reported in the preceding paper [I] that when aerobically grown Euglena gracilis is placcd under anaerobic conditions, paramylon, the reserve poly5accharide, is promptly converted into wax esters with concomitant generation of ATP, and this phenomenon has been named as the wax ester fermentation. In Euglena occurrence of four systems of fatty acid synthesis has been reported [2], namely, one involving multifunctional fatty acid synthetase in cytosol [3 -71, two acyl-carrier-protein-dependent systems in chloroplasts (one being a chain-elongation system) [4-6 , 8 -101 and one involving a fatty acid synthetase in microsomes [II, 121. In the wax ester fermentation, thesc known systems of fatty acid synthesis cannot explain the net gain of ATP, since consumption of ATP by formation of malonyl-CoA and transfer of acetyl unit from mitochondria to other subcellular sites exceeds generation of ATP in glycolysis. Existence of an unknown system of fatty acid synthesis which explain the net gain of ATP in wax ester fermentation is accordingly suggested.In the present paper we report that a malonyl-CoA independent fatty acid-synthetic system occurs in mitochondria of E. gracilis and that the system has ability to synthesize fatty acids directly from acetyl-CoA as both primer and C, donor unlike other malonyl-CoA independent systems of fatty acid synthesis in mammalian mitochondria [I 3 -161, chloroplasts of spinach [17] and Mycohacterium smegmutis [18, 191. It is also discussed that the mitochondrial system of fatty acid synthesis contributes to the wax ester fermentation.

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Section: Orgunisms and Chemicalsmentioning

confidence: 99%

Fatty acid synthesis in mitochondria of Euglena gracilis

Inui

Miyatake

Nakano

et al. 1984

European Journal of Biochemistry

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“…Glyoxylate reductase (NADP+) and glycollate dehydrogenase are localized in mitochondria to constitute the functional glycollate-glyoxylate cycle, as in the wild-type cells (Yokota & Kitaoka, 1981). Rotenone-insensitive NADPH-cytochrome c reductase, adenylate kinase, lactate dehydrogenase (cytochrome) and malate dehydrogenase (NAD+) were used as marker enzymes for outer membrane, intermembrane space, inner membrane and matrix respectively (Isegawa et al, 1984). These marker enzymes were well separated from each other and recovered in the appropriate submitochondrial fractions, although the reason for the recovery of a large amount of protein in the intermembrane-space fraction could not be elucidated.…”

Section: Resultsmentioning

confidence: 99%

“…These properties of the enzyme are advantageous to the function of the cycle. Reducing equivalents can be supplied as malate from chloroplasts, since the mitochondrial intermembrane space of Euglena also contains NADP+specific malate dehydrogenase (Isegawa et al, 1984). In addition, since the intracellular pH of Euglena is 6-6.5 (Lane & Burris, 1981), and active metabolism of glycollate during photorespiration (Yokota & Kitaoka, 1982) may cause acidification of the outside of mitochondria, the circumstances of the glyoxylate reductase-(NADP+)-localizing compartment guarantee the operation of the glycollate-glyoxylate cycle.…”

Section: Resultsmentioning

confidence: 99%

“…To avoid contamination by broken chloroplasts of the mitochondrial preparation, a streptomycinbleached mutant of E. gracilis, which had lost chloroplasts without any change in other cellular components (Buetow, 1968;Mego, 1968), was used for experiments. Cell cultivation of the bleached mutant, preparation and purification of mitochondria and submitochondrial fractionation were conducted by the method established by Isegawa et al (1984). Marker enzymes, rotenone-insensitive NADPH-cytochrome c reductase (Yokota & Kitaoka, 1979b) for the outer membrane, adenylate kinase (Bergmeyer, 1974) for the intermembrane space, lactate dehydrogenase (cytochrome) (Yokota & Kitaoka, 1979a) for the inner membrane, and malate dehydrogenase (NAD+) (Ting, 1968) for the matrix, were assayed by the methods in the cited references.…”

Section: Subfractionation Ofpurified Mitochondriamentioning

confidence: 99%

“…It serves to diminish the inhibition of ribulose bisphosphate carboxylase (Lawyer et al, 1983;Cook et al, 1984), of the C02-fixing enzyme in photosynthesis and of the photosynthetic carbonreduction cycle (Mulligan et al, 1983) by glyoxylate, and it removes excess NADPH in chloroplasts (Tolbert et al, 1970;Peterson, 1982). In Euglena gracilis, which oxidizes glycollate in mitochondria (Kitaoka et al, 1984), as do green algae (Stabenau, 1974;Bealy et al, 1976) and diatoms (Paul et al, 1975), glyoxylate reductase (NADP+) is located in mitochondria and constitutes the glycollate-glyoxylate cycle in combination with glycollate dehydrogenase (Yokota & Kitaoka, 1979b). Since NADPH is oxidized immediately after the addition of glycollate to t Present address: Institute for Virus Research,Kyoto University,Kyoto,Kyoto 606,Japan. osmotically broken mitochondria of Euglena in the presence of rotenone, it has been suggested that the glyoxylate reductase (NADP+) is located in the vicinity of glycollate dehydrogenase in mitochondria (Yokota & Kitaoka, 1979b).…”

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Purification and some properties of glyoxylate reductase (NADP+) and its functional location in mitochondria in Euglena gracilis z

Yokota¹,

Haga²,

Kitaoka³

1985

Biochemical Journal

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Euglena mitochondria contain both glyoxylate reductase (NADP+) and glycollate dehydrogenase to constitute the glycollate-glyoxylate cycle [Yokota & Kitaoka (1979) Biochem. J. 184, 189-192]. Euglena glyoxylate reductase (NADP+) was purified and its submitochondrial location was determined in order to elucidate the cycle. The purified glyoxylate reductase was homogeneous on polyacrylamide-gel electrophoresis. Difference spectra of the purified enzyme revealed that the enzyme was a flavin enzyme. The Mr of the enzyme was 82 000. The enzyme was specific for NADPH, with an apparent Km of 3.9 microM, and for glyoxylate, with an apparent Km of 45 microM. It was 30% as active with oxaloacetate as with glyoxylate. NADH and hydroxypyruvate did not support the activity at all. The optimum pH was 6.45. Submitochondrial fractionation of purified mitochondria showed that the enzyme was located in the intermembrane space and loosely bound to the outer surface of the inner membrane. These properties and the submitochondrial localization of NADPH-glyoxylate reductase facilitate the operation of the glycollate-glyoxylate cycle in combination with glycollate dehydrogenase, which is tightly bound to the inner membrane of Euglena mitochondria.

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Submitochondrial Location and Some Properties of NAD⁺- and NADP⁺-Linked Malate Dehydrogenase inEuglena

Cited by 2 publications

References 4 publications

Fatty acid synthesis in mitochondria of Euglena gracilis

Fatty acid synthesis in mitochondria of Euglena gracilis

Purification and some properties of glyoxylate reductase (NADP+) and its functional location in mitochondria in Euglena gracilis z

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